A central tenant of tissue engineering is that cells should be able to recapitulate full functional tissue capability when placed within an appropriate architecture or scaffold. The aim of this study was to examine the effect of three-dimensional (3D) architecture on the differentiated phenotype of human smooth muscle cells derived from the stroma of the lower urinary tract. Stromal cell cultures were established from surgical specimens and the differentiated smooth muscle cell phenotype was monitored by gene expression, immunofluorescence and immunoblotting. Expression of contractile proteins, including smooth muscle myosin and smoothelin, was lost by cultures grown on two-dimensional (2D) tissue culture polystyrene, but was regained to some extent by the removal of serum and by the addition of TGFβ1. Stromal cells were seeded onto plasma-coated electrospun polystyrene scaffolds to examine the influence of 3D architecture on smooth muscle cell phenotype, but differentiation was inhibited by serum proteins that adsorbed non-specifically onto the large surface area of the scaffold. Stromal cells failed to adhere to the scaffold in serum-free conditions, but laminin pre-coating of the scaffold prevented serum adsorption and promoted cell attachment and differentiation. The study highlights how non-specific factors, such as serum adsorption, may confound the development of materials for tissue engineering.